INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 190-200 Vol. 38. No. 2 0020-77 13/88/020190-11$02.OO/O Copyright 0 1988, International Union of Microbiological Societies Phylogenetic Study of the Genus Campylobacter LOUIS M. THOMPSON 111,’ ROBERT M. SMIBERT,2 JOHN L. JOHNSON,2 AND NOEL R. KRIEG1* Microbiology and Immunology Section, Department of Biology,’ and Department of Anaerobic Microbiology,2 Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 The phylogenetic relationships of all species in the genus Cantpylobacter, Wolinella succinogenes, and other gram-negative bacteria were determined by comparison of partial 16s ribosomal ribonucleic acid sequences. The results of this study indicate that species now recognized in the genus Campylobacter make up three separate ribosomal ribonucleic acid sequence homology groups. Homology group I contains the following true Campylobacter species: Campylobacterfetus (type species), Campylobacter coli, Campylobacter jejuni, Campylo- bacter laridis, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, and ‘‘Campylobacter upsaliensis” (CNW strains). “Campylobacter cinaedi,” ‘Campylobacter fennelliue,” Campylobacter pylori, and W.succinogenes constitute homology group 11. Homology group I11 contains Campylobacter ctyaerophiza and Campylobacter nitrofigilis. We consider the three homology groups to represent separate genera. However, at present, easily determinable phenotypic characteristics needed to clearly differentiate them are not apparent. The three homology groups are only distantly related to representatives of the alpha, beta, and gamma branches of the purple bacteria, indicating that these bacteria do not belong to any previously defined branch of this phylum. Campylobacters have been recognized for many years as the CNW strains represent a distinct species (36). Campylo- important agents of reproductive diseases in sheep and bacter cryaerophila (sic) is an aerobic species that causes cattle, and some of the species have also been found to cause abortions in pigs, cattle, horses, and sheep and can occa- diseases in humans. The type species of the genus, Cam- sionally be isolated from human infections (17, 27), whereas pylobacter fetus, is divided into two subspecies; C. fetus Campylobacter nitrofigilis is a microaerophilic, NaC1-re- subsp. venerealis causes sexually transmitted abortions in quiring, nitrogen-fixing bacterium found in the roots of salt cattle, and C. fetus subsp. fetus causes orally transmitted marsh grasses (25). sporadic abortions in cattle and sheep and blood infections in The classification of the genus Campylobacter has always humans (39). Campylobacter hyointestinalis has been iso- been somewhat difficult. Because these organisms do not lated from pigs with proliferative ileitis (8) and occasionally catabolize carbohydrates and are inert with regard to most from homosexual males with proctitis (5). Campylobacter traditional biochemical tests used for the identification of mucosalis has been isolated from lesions of porcine intesti- bacteria, only a relatively small number of tests are available nal adenomatosis (8, 20, 39). Campylobacter jejuni is part of for the identification and classification of campylobacters the normal intestinal flora of cattle, sheep, dogs, cats, (35, 36). Consequently, classification of campylobacters poultry, and other animals (39), and it is a major cause of based on only a few biochemical and physiological tests bacterial gastroenteritis in humans (3). Campylobacter coli is gives few means to accurately differentiate species. part of the normal flora of pigs and poultry and can also Although DNA homology studies have shown that the Campylobacter laridis cause diarrhea in humans. occurs in current Campylobacter species are distinct from one another the intestines of sea gulls, humans, dogs, and horses and (1, 2, 9, 11, 21, 30, 31, 35, 36, 47), they have not answered occasionally causes blood infections and diarrhea in humans the question of whether these species are sufficiently related (15, 26, 38, 45). Campylobacter concisus has been isolated from the oral cavities of people with periodontal disease (42). to justify classification within a single genus. Comparison of Campylobacter sputorum, which is divided into three bio- 16s ribosomal ribonucleic acid (rRNA) sequences for phy- vars, biovars sputorum, bubulus, and fecalis, occurs as part logenetic analysis has proven to be a powerful tool for of the normal flora of human mouths, bovine genitalia, and accurate classification of microorganisms above the level of sheep feces, respectively (36). Campylobacter pylori is a species (7, 29, 40, 41, 49, 50). The recent development of a probable causative agent of gastric and duodenal ulcers and technique which facilitates the rapid generation of partial chronic gastritis in humans (22, 23). “Campylobacter 16s rRNA sequences has allowed researchers to determine cinaedi” and “Campylobacter fennelliae” have been asso- the phylogenetic relationships among bacteria (18). Using ciated with proctitis, proctocolitis, enteritis, and bacteremia this technique, Romaniuk et al. (34) concluded that C.pylori in homosexual men (4,6,28,46).CNW (catalase-negative or is not related at the genus level to C.jejuni, C. coli, C.fetus weakly catalase-positive) strains of Campylobacter have subsp.fetus, C. laridis, or C. sputorum biovar sputorum and been isolated from healthy and diarrheic dogs and cats (37; that these latter species represent the true genus Campylo- C. J. Gebhart, G. E. Ward, and L. A. Finsmith, Abstr. bacter. C. pylori was more closely related to Wolinella Annu. Meet. Am. SOC. Microbiol. 1984, C55, p. 245); the succinogenes than to the other campylobacters. Using data name “Campylobacter upsaliensis” has been suggested for based on partial 16s rRNA sequences, Lau et al. (19) these organisms (K. Sandstedt and J. Ursing, XIV Int. reported that C. jejuni, C. coli, and C. laridis are very Congr. Microbiol., 1986, abstr. no. P.B8-17). Deoxyribonu- closely related, that the C. jejuni-C. coli-C. laridis group, C. cleic acid (DNA) reassociation experiments have shown that fetus, C. sputorum biovar fecalis, and C. pylori are all distinct from one another, and that W. succinogenes is related to the campylobacters. More recently, Paster and * Corresponding author. Dewhirst (32) confirmed the relationship between W. succi- 190 VOL. 38, 1988 PHYLOGENETIC STUDY OF CAMPYLOBACTER 191 TABLE 1. Strains of Cutnp~lohucrerspecies used in this study isolated by a modification of a previously described proce- Species“ Strain“ dure (14. 16). Lysates of cells disrupted with a French pressure cell were extracted with a phenol-cresol solution; Catalase-positive campylobacters 16s rRNA was selectively precipitated from the soluble C. coli .................................................. ATCC 33559’ 7.5 C. jejirtti ................................................ ATCC 33560’ ribonucleic acid (RNA) and DNA by adding cold (-20°C) C. ferrrs subsp. .fitus ............................... ATCC 27374’ M ammonium acetate. The rRNA was stored at -20°C in a C. .fetzr.s subsp. venereulis ....................... .ATCC 19438’ buffer consisting of 0.15 M NaCI, 0.01 M sodium ethylene- C. hyointestiticilis ................................... .80-4577-4 (= ATCC diaminetetraacetate, 1.0 mM N-2-hydroxyethylpiperazine- 35217Ih N’-2-ethanesulfonic acid (HEPES buffer), and 1.0% sodium C. luridis .............................................. .NCTC 11352T dodecyl sulfate. The sodium dodecyl sulfate was included to C. cryueropttila ..................................... .NCTC lltWT inhibit the activity of ribonucleases; its omission resulted in C. nitrojgilis.. ....................................... .ATCC 33309T RNA degradation even at -20°C. “C. cinaedi” ........................................ .ATCC 35683‘ Synthesis and purification of oligonucleotide primers. Prim- “C. fennelliue” ...................................... ATCC 35684‘ C. py/ori ............................................... NCTC 11637T ers complementary to conserved regions of the 16s rRNA Cataiase-negative campylobacters molecule were prepared by using a DNA synthesizer (model C. mrrcoscilis .......................................... NCTC 11000’ 381A: Applied Biosystems, Foster City, Calif.). The five C. conc.isu.s.. .......................................... VPI 13086T primers used in this study were complementary to the C. sputoriim biovar sputorum ...................VPI S-17 (= ATCC following regions of Eschericlzin cdi 16s rRNA (5’ to 3’): 35980)“ positions 321 to 340,519 to 536,907 to 926,1220 to 1239, and CNW (“C. rrpsaliensis”) .......................... CG-1” 1388 to 1407. Crude oligonucleotide preparations were puri- ‘‘ ATCC. American Type Culture Collection, Rockville. Md.: NCTC. fied by thin-layer chromatography (Brian Reid, personal National Collection of Type Cultures. London. England: VPI. Virginia communication). diluted to a concentration of 0.1 mg/ml Polytechnic Institute and State University. Blacksburg: CNW. catalase neg- with TE buffer [lo mM tris(hydroxyrnethy1)aminomethane ative or weakly positive: T = type strain. base and 0.1 mM ethylenediaminetetraacetate, pH 8.01, and ” Type strain proposed by Gebhart et al. (8). ‘ Type strain proposed by Totten et al. (46). stored at -20°C. ‘‘ Neotype strain proposed by Roop et al. (36). Preparation of RNA for sequencing. The sodium dodecyl ‘’ Reference strain C.